Padh et al., IJPSR, 2018; Vol. 9(8): 3355-3359.

E-ISSN: 0975-8232; P-ISSN: 2320-5148

IJPSR (2018), Volume 9, Issue 8 (Research Article)

Received on 25 November, 2017; received in revised form, 11 February, 2018; accepted, 04 March, 2018; published 01 August, 2018

DEVELOPMENT AND VALIDATION OF Q-ABSORBANCE RATIO SPECTROPHOTO-

METRIC METHOD FOR SIMULTANEOUS ESTIMATION OF MANGIFERIN AND BERBERIN
HCl IN BULK AND SYNTHETIC MIXTURE
Harish Padh, Shraddha Parmar * and Bhavna Patel
Department of Pharmaceutical Sciences, Sardar Patel University, Vallabh Vidyanagar - 388120, Gujarat,
India.
Keywords: ABSTRACT: There are so many promising plant based chemical constituents
are present which act as alternative therapy for the control of diabetes. But due
Mangiferin, Berberin HCl,
Simultaneous equation method, to lack of its proper quality control parameters they are not widely used. The US
ICH guidelines, Quality control FDA patent is approved for fixed dosage combination of Mangiferin (MF) and
Correspondence to Author: Berberin HCl (BER) as antidiabetic herbal formulation. Absorbance ratio
Shraddha Parmar method uses the ratio of absorbances at two selected wavelengths, one which is
an iso-absorptive point and other being the λmax of one of the two components.
Assistant Professor, From the overlay spectra of two phytomarkers, it is evident that MF and BER
Department of Pharmaceutical show an iso-absorptive point at 317 nm. The second wavelength used is 257 nm,
Sciences, Sardar Patel University which is the λmax of MF. The drug response with respect to absorbance was
Vallabh Vidyanagar - 388120 linear over the concentration range 5 - 30 μg/ml for MF and 10 - 60 μg/ml for
Gujarat, India.
BER. The percentage recovery of MF and BER as found to be 100.00% and
E-mail: shraddhajparmar@gmail.com 100.07% respectively. The method can be successfully employed for the
simultaneous determination of MF and BER in pharmaceutical formulations.
The developed method is validated as per ICH guideline Q2 (R1).
INTRODUCTION: The number of drugs and drug It is also revealed that no UV spectroscopic method
formulations introduced into the market has been has been reported in the literature review for the
increasing at an alarming rate. These drugs or combination formulation of Mangiferin and
formulations may be either new entities or partial Berberin HCl. The condition thus, provides the
structural modification of the existing ones or novel scope of development of accurate, sensitive,
dosage forms. The US FDA US 7867979 B2 patent reproducible and simple Spectroscopic method
is approved for fixed dosage combination of MF capable of estimating both the drugs from the
and BER as antidiabetic herbal formulation 1 - 2. formulation simultaneously 3, 4, 5, 6, 7, 8, 9.
The literature review suggested that various HPLC,
HPTLC and UV-visible Spectrophotometric Experimental:
methods have been reported for estimation of MF Apparatus and Instrument: A Double beam UV-
and BER individually or in combination with other Visible Spectrophotometer (Shimadzu, model
drugs from pharmaceutical dosage form. pharmaspec 1800) having two matched quartz cells
with 1 cm light path and Electronic analytical
QUICK RESPONSE CODE balance, (Shimadzu AUX-220) was used. Corning
DOI:
10.13040/IJPSR.0975-8232.9(8).3355-59
volumetric flasks, pipettes of borosilicate glasses
were used in the study.
Article can be accessed online on: Spectrophotometric Conditions:
www.ijpsr.com
1. Mode: Spectrum
DOI link: http://dx.doi.org/10.13040/IJPSR.0975-8232.9(8).3355-59
2. Scan speed: Fast

International Journal of Pharmaceutical Sciences and Research 3355

Padh et al., IJPSR, 2018; Vol. 9(8): 3355-3359.
3. Wavelength range: 400 - 200 nm
4. Absorbance scale: 0.00A - 2.00A
5. Initial base line correction: Methanol
Chemicals and Reagents: MF reference standard
was purchased from Sigma Aldrich. BER reference
standard was given as gift sample by Enovate life
Mumbai.
Preparation of Standard Solutions: To Prepare
standard solution of MF (100 μg/ml) and BER
(1000 μg/ml), accurately weigh 25 mg of each
drugs were transferred in two different 100 and 10
ml volumetric flasks respectively, dissolve and
diluted up to mark with methanol, from these stock
solutions, 5 ml and 1 ml aliquots of MF and BER
respectively were transferred in two different 10 ml
volumetric flasks and were diluted up to mark with
methanol to get working standard solution having
concentration of MF of 50 μg/ml and BER of 100
μg/ml.
Methodology: Absorbance ratio method uses the
ratio of absorbances at two selected wavelengths,
one which is an iso-absorptive point and other
being the λmax of one of the two components. From
the overlay spectra of two drugs, it is evident that
MF and BER show an iso-absorptive point at 317
nm. The second wavelength used is 257 nm, which
is the λmax of MF. Working standards were
prepared in methanol and the absorbances at 317
nm (iso-absorptive point) and 257 nm (λmax of MF)
were measured and absorptivity coefficients were
calculated using calibration curve. The
concentration of two drugs in the mixture can be
calculated using following equations.
CX = [(QM – QY) / (QX -QY)] × A1 / ax1 .... (1)
CY = [(QM – QX) / (QY -QX)] × A1 /ay1 … (2)
Where, A1 and A2 are absorbances of mixture at
257 nm and 317 nm; ax1 and ay1 are absorptivities
of MF and BER at 257 nm; ax2 and ay2 are
absorptivities of MF and BER respectively at 317
nm;
QM = A2 / A1, QX = ax2 / ax1, QY = ay2 / ay1
Calibration Curve for MF and BER: To check
linearity of the method, working standard solution
having concentration in range of 5 - 30 μg/ml of
International Journal of Pharmaceutical Sciences and Research
E-ISSN: 0975-8232; P-ISSN: 2320-5148
MF and 10 - 60 μg/ml of BER were prepared from
the standard stock solutions of both drugs. The
absorbance was measured at 257 nm (λmax of MF)
and at 317 nm (λmax of BER). Calibration curves
were constructed by plotting absorbance vs.
concentration.
Preparation of Sample Solution from
Laboratory Prepared Synthetic Mixture:
Synthetic mixture of MF (25 mg) and BER (75 mg)
was prepared by using common excipients like
Corn Starch (50 mg), Lactose (113 mg) and
Magnesium Stearate (2 mg) per tablet. Tablet
powder was prepared by calculating formula for 10
Tablets having label claim for MF and BER 25 mg
and 75 mg respectively. From this mixture, powder
equivalent to 45 mg BER was dissolved in 250 ml
methanol and then sonicated for 15 min. and
filtered through Whatman filter paper. From this
solution, 2.5 ml aliquot was taken in 10 ml
volumetric flask and diluted up to the mark with
methanol to make final concentration of MF and
BER, 15 μg/ml and 45 μg/ml, respectively which
was used for assay.
RESULTS AND DISCUSSION:
Method Development: For this measurement, the
solutions of MF and BER were prepared separately
in methanol at a concentration of 15 μg/ml and 45
μg/ml respectively. They were scanned in the
wavelength range of 200 - 400 nm. From the
overlay spectra of two drugs, it is evident that MF
and BER show an iso-absorptive point at 317 nm.
MF showed 241 nm, 257 nm, 317 nm, 366 nm λmax
from which 257 nm was selected as λmax of MF in
this method due to good linearity Fig. 1.
Validation of the Proposed Method:
Linearity: Linear correlation was obtained
between absorbance vs. concentration of MF and
BER in the concentration ranges of 5 - 30 μg/ml
and 10 - 60 μg/ml respectively and is shown in
overlain chromatogram of MF and BER, Fig. 2 and
3 respectively.
Calibration curve data of MF and BER shown in
Table 1 and 2. Regression parameters are
mentioned in Table 3 and the linearity spectra and
calibration curves of these two drugs at 257 nm and
317 nm are shown in Fig. 4, 5 and 6, 7
respectively.
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Padh et al., IJPSR, 2018; Vol. 9(8): 3355-3359. E-ISSN: 0975-8232; P-ISSN: 2320-5148

FIG. 1: ZERO ORDER OVERLAIN SPECTRA OF MF (10 μg/ml), BER (45 μg/ml) AND COMBINATION (10 μg/ml
MF AND 45 μg/ml BER)

A B
FIG. 2: (A) OVERLAIN SPECTRA OF MF (5 - 30 μg/ml) IN METHANOL (B) OVERLAIN SPECTRA OF BER (10 -
60 μg/ml) IN METHANOL
TABLE 1: LINEARITY DATA FOR MF
S. no. Concentration of MF At Wavelength 257 nm At Wavelength 317 nm
(μg/ml) Absorbance (Mean ± SD) % RSD Absorbance (Mean ± SD) % RSD
1 5 0.224 ± 0.004 1.61 0.221 ± 0.003 0.44
2 10 0.391 ± 0.002 0.62 0.293 ± 0.004 0.94
3 15 0.555 ± 0.002 0.59 0.345 ± 0.004 0.34
4 20 0.687 ± 0.003 0.26 0.434 ± 0.002 0.55
5 25 0.745 ± 0.004 0.36 0.500 ± 0.002 0.72
6 30 0.895 ± 0.003 0.34 0.586 ± 0.004 0.94

TABLE 2: LINEARITY DATA FOR BER

S. no. Concentration of BER At Wavelength 257 nm At Wavelength 317 nm
(μg/ml) Absorbance (Mean ± SD) % RSD Absorbance (Mean ± SD) % RSD
1 10 0.305 ± 0.002 1.00 0.221 ± 0.005 1.05
2 20 0.415 ± 0.004 0.33 0.290 ± 0.004 1.01
3 30 0.525 ± 0.003 1.22 0.355 ± 0.004 0.75
4 40 0.637 ± 0.002 0.26 0.430 ± 0.002 0.45
5 50 0.745 ± 0.003 1.36 0.500 ± 0.005 0.71
6 60 0.860 ± 0.002 0.94 0.590 ± 0.005 1.40

TABLE 3: LINEARITY DATA

Parameter MF at 257 nm MF at 317 nm BER at 257 nm BER at 317 nm
Linearity range (μg/ml) 5 - 30 5 - 30 10 - 60 10 - 60
Regression equation y = 0.0252x + 0.1487 y = 0.014x + 0.142 y = 0.0111x + 0.1931 y = 0.007x + 0.141
Correlation coefficient (r2) 0.9991 0.9980 1.000 0.9980
y-intercept 0.1487 0.142 0.1931 0.141
Standard deviation of slope 0.0056 0.0098 0.0054 0.0066

Accuracy: Accuracy of the method was confirmed
by recovery study from marketed formulation at
three levels of standard additions (80%, 100% and
120%). Percentage recovery for MF was in the
range of 99.99 - 100.07%, while for BER, it was
found to be in range of 99.84 - 101.10%. The
results are shown in Table 4 and 5. Recovery
greater than 98% with low SD justifies the
accuracy of the method.

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Padh et al., IJPSR, 2018; Vol. 9(8): 3355-3359. E-ISSN: 0975-8232; P-ISSN: 2320-5148

FIG. 4: CALIBRATION CURVE OF MF FIG. 5: CALIBRATION CURVE OF MF

(5 - 30 μg/ml) AT 257 nm (5 - 30 μg/ml) AT 317 nm

FIG. 6: CALIBRATION CURVE OF BER FIG. 7: CALIBRATION CURVE OF BER

(10 - 60 μg/ml) AT 257nm (10 - 60 μg/ml) AT 257nm

TABLE 4: RECOVERY DATA FOR MF found to be 1.12% and 1.02% at 257 nm and 317
% Conc. of MF Conc. of MF % Recovery of %
Level in sample recovered MF (% RSD
nm, respectively for MF and 1.03 and 0.90% at 257
(μg/ml) (μg/ml) Recovery ± SD) nm and 317 nm respectively for BER. % R.S.D was
80% 12 11.90 99.99 ± 1.92 1.92 less than 2% complied with the standard limits.
12 11.67
12 12.10 TABLE 6: REPEATABILITY DATA FOR MF AND BER
100% 15 14.02 100.00 ± 0.65 0.65 Drug Conc. Abs. (Mean ± % Abs. (Mean ± %
15 15.98 (μg/ml) SD) at 257 RSD SD) at 317 RSD
15 15.10 nm nm
120% 18 18.09 100.07 ± 0.49 0.49 MF 15 0.555 ± 1.12 0.343 ± 1.02
18 18.00 0.008 0.006
18 17.95 BER 45 0.759 ± 1.03 0.353 ± 0.90
0.007 0.004
TABLE 5: RECOVERY DATA FOR BER
% Conc. of BER Conc. of BER % Recovery of % Intra Day and Inter Day Precision (n = 3): The
Level in sample recovered BER (%) RSD
(μg/ml) (μg/ml) data for intraday precision for MF and BER is
80% 36 36.11 101.83 ± 1.19 1.18 shown in Table 7. The % R.S.D. for Intraday
36 36.79 precision was found to be 0.22 - 0.40% for MF and
36 36.00
100% 45 45.02 100.07 ± 0.14 1.12 0.34 - 0.56% for BER at 257 nm respectively and
45 44.98 0.45 - 1.26% for MF and 0.37 - 0.59% for BER at
45 45.10 317 nm respectively. The data for intraday
120% 54 54.09 99.84 ± 0.30 0.30
54 53.77 precision for MF and BER is shown in Table 8.
54 53.88 The % R.S.D. for interday precision was found to
be 0.96 - 1.75% for MF and 1.58 - 1.89% for BER
Precision: at 257 nm respectively and 0.96 - 1.01% for MF
Repeatability (n = 6): The repeatability was and 0.89 - 1.89% for BER at 317 nm respectively.
checked by scanning and measurement of the
responses of solutions of MF (5 - 30 μg/ml) and LOD and LOQ: The LOD was calculated by
BER (10 - 60 μg/ml) without changing the standard deviation of response and was found to be
parameters of the proposed method. The procedure 2.33 μg/ml and 5.22 μg/ml for MF and BER
was repeated six times and % RSD was calculated. respectively at 257nm, be 2.30 μg/ml and 4.32
The data for repeatability for combined solution of μg/ml for MF and BER respectively at 317 nm. The
MF and BER is presented in Table 6. % R.S.D was LOQ was calculated by standard deviation of

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Padh et al., IJPSR, 2018; Vol. 9(8): 3355-3359. E-ISSN: 0975-8232; P-ISSN: 2320-5148

response and was found to be 3.91 μg/ml and 6.69 3.22 μg/ml and 7.22 μg/ml for MF and BER
μg/ml for MF and BER respectively at 257 nm, be respectively at 317 nm.
TABLE 7: INTRADAY PRECISION DATA FOR MF AND BER
S. no. Drug Concentration Absorbance % RSD Absorbance % RSD
(μg/ml) (Mean ± SD) at 257 nm (Mean ± SD) 317 nm
1 MF 10 0.391 ± 0.002 0.22 0.520 ± 0.005 0.45
2 15 0.555 ± 0.002 0.23 0.630 ± 0.005 0.67
3 20 0.687 ± 0.003 0.40 0.756 ± 0.008 1.26
1 BER 30 0.565 ± 0.004 0.44 0.522 ± 0.002 0.37
2 40 0.672 ± 0.003 0.34 0.635 ± 0.005 0.59
3 50 0.745 ± 0.004 0.56 0.743 ± 0.005 0.54

TABLE 8: INTERDAY PRECISION DATA FOR MF AND BER

S. no. Drug Concentration (μg/ml) Absorbance % RSD Absorbance % RSD
(Mean ± SD) at 257 nm (Mean ± SD) at 317 nm
1 MF 5 0.391 ± 0.005 1.75 0.521 ± 0.006 1.00
2 10 0.545 ± 0.007 1.02 0.631 ± 0.003 1.01
3 15 0.685 ± 0.008 0.96 0.755 ± 0.008 0.96
1 BER 30 0.563 ± 0.008 1.58 0.523 ± 0.008 0.89
2 40 0.672 ± 0.007 1.65 0.636 ± 0.007 1.36
3 50 0.744 ± 0.005 1.89 0.742 ± 0.007 1.85

Analysis of Synthetic Mixture: Here, 15 μg/ml CONFLICT OF INTEREST: Nil

solution of MF and 45 μg/ml solution of BER
synthetic mixture were prepared in triplicate
manner and analyzed. The assay was carried out as
per regression equation. The result of assay is
shown in Table 9.
TABLE 9: ASSAY OF SYNTHETIC MIXTURE
Drug Amount of drug
(mg)
MF 25
BER 75
CONCLUSION: A new, simple, accurate, and
precise UV spectroscopic method was developed
for the simultaneous estimation of MF and BER in
bulk drugs and in the presence of tablet excipients.
The recovery studies suggested non-interference of
formulation excipients in the estimation. Hence, the
proposed method can be used for the quality
control of the cited drugs and can be extended for
routine analysis of the drugs in their
pharmaceutical dosage forms.
ACKNOWLEDGEMENT: The authors wish to
thank Enovate life Mumbai and PRS Herbal
Haryana for providing Berberine HCl as gift
sample. We would like to acknowledge Sardar
Patel University, Vallabh Vidyanagar for providing
seed grant to support this research work.
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How to cite this article:

Padh H, Parmar S and Patel B and: Development and validation of q-absorbance ratio spectrophotometric method for simultaneous estimation of
mangiferin and berberin HCl in bulk and synthetic mixture. Int J Pharm Sci Res 2018; 9(8): 3355-59. doi: 10.13040/IJPSR.0975-8232.9(8).3355-59.
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